CN101121925A

CN101121925A - Strain for producing arginine deiminase and application thereof

Info

Publication number: CN101121925A
Application number: CNA200710107822XA
Authority: CN
Inventors: 孙志浩; 郑璞; 倪晔; 刘咏梅; 刘宇鹏
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2007-05-17
Filing date: 2007-05-17
Publication date: 2008-02-13
Anticipated expiration: 2027-05-17
Also published as: CN100584940C; WO2008141523A1; WO2008141523A8

Abstract

The invention discloses a method to produce ADI strain by screening and the application of the method, pertaining to the field of bioengineering technology. The invention particularly relates to a rapid method to screen ADI strain and produce Pseudomonas plecoglossicida (CGMCC No.2039) of ADI, as well as a method to produce ADI by using the microorganism. Under an aerobic condition and an environment maintained at pH6.5-8.0, enzyme activity can achieve 1.6U/mL fermentation broth after 20-24h fermentation culture. ADI produced with such microorganism has very strong inhabitation agaisnt liver cancer cell strain (HepG2). ADI genes can be obtained from the chromosome DNA of such microorganism, and the gene order can be achieved.

Description

The bacterial classification and the application thereof of arginine deiminase produced in one strain

Technical field

The bacterial classification and the application thereof of arginine deiminase produced in one strain, belongs to technical field of bioengineering.Be specifically related to bacterial classification and method, and keep the application aspect medical of enzyme that the strain of going down to posterity, the variant of this active this bacterium and the strain of deriving produce with a kind of distortion pseudomonas (Pseudomonas plecoglossicida) CGMCC.2039 fermentative production arginine deiminase (ADI).

Background technology

Arginine deiminase (Arginine deiminase) is called for short ADI.A lot of microorganisms utilize ADI hydrolysis arginine to obtain energy, and in the arginine deiminase pathways metabolism, the first step reaction is ADI hydrolysis arginine and generates citrulline and ammonia.Arginine is a kind of non-essential amino acid for the mankind.Normal nucleus tissue can pass through citrulline, and is synthetic under urea cyclophorase arginine-succsinic acid (ASS) synthetic enzyme and arginine-amber acidifying enzyme effect.Some do not express ASS by quasi-cancer cell, thereby can not be from the synthetic arginine of citrulline.Therefore, arginine defective type cancer cells might fundamentally be eliminated or control to the arginine degrading enzyme.

Nineteen thirty, Gilroy proves for the first time, uses to be rich in arginic forage feed mouse, compares with the mouse of using common forage feed, and its tumor proliferation quickens and tumour is individual increases.The arginine Degradation that discovery mycoplasmas such as Schimke produce is caused that by a kind of novel enzyme be called ADI, it can make arginine be converted into citrulline and ammonia, rather than makes arginine be converted into ornithine and urea as arginase.

1992, people such as Takaku separated from mycoplasma for the first time and obtain ADI and find that this kind of enzyme in vivo and the external activity that all exists.But need heavy dose of frequent being input in the body to reach effective anticancer effect.1999, under the effect of the lower concentration arginine deiminase that Hong Gong etc. extracts, the cell cycle can be stopped at G from Mycoplasma arginini ₁With the S phase, to suppress the propagation of various kinds of cell; Under the effect of high density arginine deiminase, can cause concurrent apoptosis.They studies have shown that the arginine desaminase not only passes through to arginic degraded, also by comprising the related mechanism of cell cycle and apoptotic signal for the inhibition of cell proliferation.

2002, the Charles Mark Ensor of Kentucky, USA university, Deng, (ADI-PEG) is used for experiment on mice with ADI-PEG, HPLC detects and finds that ADI only transforms arginine, proved that simultaneously melanoma and hepatoma lack smart ammonia (base) succsinic acid synthetic enzyme mRNA, not synthetic arginic ability, ADI has the inhibition effect to arginine defective type cancer cells.2004, they were used for clinical experiment with ADI-SS PEG, had infected in patient's blood plasma of HCC and had detected less than arginic existence, and for some patients, this therapy is effective.

Well-known also is the Radix Asparagi amidase that uses maximum inhibition tumor growths clinically, and this medicine has obtained successful application when treatment acute lymphoblastic leukemia and associated malignancies.But, follow the l-asparagine enzyme treatment can produce, coagulopathy, serious side effects such as liver and pancreas poisoning as anaphylactic shock.Asparaginase is at the degraded glutamine of also degrading in the l-asparagine.The anticancer vigor that studies show that this enzyme is because its degraded l-asparagine, and its some side effects are because the time, show that the side effect of arginine desaminase is less, it has better application clinically and is worth.When using the aspartoyl enzyme to produce the untoward reaction generation, as the substitute of aspartoyl enzyme.

In the present report both domestic and external, that produces the ADI enzyme has Pseudomonas aeruginosa (Pseudomonas aeruginosa) (A.Galkin, J.Biol.Chem..279,14001-14008,2004), pseudomonas putida (Pseudomonas putida) (T.Kakimoto, Appl.Microbiol., .1971, .992-999), and mycoplasma (Mycoplasma homnisi) (H.Gong Biochem.Biophy.Res.Comm.261,10-141999); Mycoplasma (Mycoplasma arginini) (C.M.Ensor Cancer Research62,5443-5450,, 2002; F.Izzo J.Clinical Oncology, 22,2004) etc.1971, the people such as T Kakimoto of Japan filtered out pseudomonas putida as the bacterial classification of producing citrulline, and by 30 ℃ of shake flask fermentation productions, the ADI enzyme work of this bacterial classification reaches 9.2U/ml.J.B.Jones (1981) at first uses the ADI that purifies from Pseudomonas pudita as a kind of anticancer preparation.Although this kind of enzyme at extracorporeal suppression tumor cell, is not observed tangible activity in vivo.1992, people such as H.Takaku separated from mycoplasma for the first time and obtain ADI and find that this kind of enzyme in vivo and the external activity (H.Takaku, et al.J.Cancer, 51:244-249,1992.) that all exists.Subsequently, a lot of researchs are produced ADI relevant for mycoplasma, and studies have shown that this bacterium produces ADI anticancer effect is arranged.But Pseudomonas aeruginosa is pathogenic bacterium, and pseudomonas putida is a conditioned pathogen, and mycoplasma is that very highly toxic pathogenic agent is arranged.Be not the report of human pathogenic bacterium at present, the report of distortion pseudomonas fermentative production ADI is not arranged as yet yet about distortion pseudomonas (Pseudomonas plecoglossicida).

Summary of the invention

The purpose of this invention is to provide bacterial strain and method that a kind of microbial fermentation produces arginine deiminase, this bacterial strain be a strain new bacterial strain that can effectively produce arginine deiminase, we to its identify, enzymatic production and Anticancer Activities.

It below is the detailed description of technical solution of the present invention.

The screening of microorganism strains and evaluation:

In the present invention, set up the model of a screening product arginine deiminase bacterial strain rapidly and efficiently.According to the characteristics that the bacterial strain catalysis arginine that produces arginine deiminase produces citrulline and ammonia, designing with the arginine is the selectivity flat board of carbon source, filters out and utilizes arginic bacterial strain.Because arginine deiminase catalysis arginine produces ammonia, therefore in containing the acid flat board of phenolsulfonphthalein developer, can produce red variable color circle.

The present invention is from 5 parts of soil samplings such as Wuxi Hui Shan, Xihui Park, Wuxi, canal, Hangzhoupro, capital section, 2 parts in plant materials sample, 2 parts of water samples, 9 duplicate samples altogether.Got back rapid strain screening within 12h.The concrete grammar of screening is: sample behind the enrichment culture 24h, is coated with the screening culture medium flat board again in enrichment medium.Through plate screening, be divided into from obtaining 54 strains and produce the bacterial strain of arginine deiminase.Further carry out multiple sieve, measure the ADI enzyme with Diacetylmonoxime thiosemicarbazide method and live, finishing screen is selected aimed strain SW-P1.

The bacterial strain SW-P1 of the product arginine deiminase that the present invention's screening obtains, identify through 16S rRNA genetic testing with by " common bacteria system identification handbook " and " uncle Jie Shi Bacteria Identification handbook (the 8th edition) " Physiology and biochemistry, think the distortion pseudomonas (Pseudomonasplecoglossicida) that belongs to pseudomonadaceae (Pseudomonadaceae) Rhodopseudomonas (Pseudomonas).The cell of this bacterium is shaft-like, and is movable, and no gemma, electron microscopic observation have a lophotricha, and what have does not have a flagellum.Gram-negative, aerobic.This bacterial strain is rounded, faint yellow, opaque at the bacterium colony of LB substratum 24h, smooth surface, neat in edge, the about 1mm of diameter; Also be faint yellow at the fermention medium bacterium colony, diameter is about 1.5mm.

The preservation of biological sample material: distortion pseudomonas (Pseudomonas plecoglossicida) the bacterial strain SW-P1 that the present invention's screening obtains was kept at Zhong Guan-cun, BeiJing, China China Committee for Culture Collection of Microorganisms common micro-organisms preservation center, preserving number CGMCC No.2039 on May 11st, 2007.

The cultivation of microorganism strains:

Consisting of of slant medium: yeast extract paste 1-5g/L, peptone 1-5g/L,, extractum carnis 1-5g/L, NaCl 1-10g/L, agar 15-20g/L, pH 6.0-7.5.

Consisting of of seed and fermention medium: glucose 5-20g/L, yeast extract paste 1-5g/L, peptone 1-5g/L, L-arginine monohydrochloride 5-20g/L, Na ₂HPO ₄12H ₂O 6-15g/L, NaH ₂PO ₄2H ₂O 1-5g/L, (NH ₃) ₂HPO ₄1-5g, pH6.5-7.5;

The sterilising temp of substratum is 115-121 ℃, 15min.Packing after glucose is sterilized separately.

Make the inclined-plane according to a conventional method,, 28-35 ℃, cultivated 20-40 hour with CGMCC 2039 inoculation that screening obtains.In seed culture medium, the bottled liquid 40-100ml of 250ml triangle cultivated 18-28 hour down in 28-35 ℃ of aerobic condition with the inoculation on the inclined-plane.Inoculum size by 1%-10% inserts fermention medium with cultured seed, and culture temperature 28-35 ℃, aerobic cultivation 20-40 hour.

ADI enzyme activity determination method:

The ADI enzyme activity determination is to be that the amount of the citrulline that generates in the conversion fluid of substrate is determined by detecting with the L-arginine monohydrochloride.Concrete grammar: get the 5mL fermented liquid, centrifugal collection thalline, the physiological saline washed twice, add 5ml 0.1%CTAB, leave standstill 10min, centrifugal collecting precipitation in 37 ℃ of incubators, add 5mL 0.1mol/L L-arginine monohydrochloride-phosphoric acid buffer pH 6.0, mixing leaves standstill 0.5h in 37 ℃ of incubators and transforms the ice bath termination reaction.Add nitration mixture 3mL in the 10mL colorimetric cylinder successively, Diacetylmonoxime thiosemicarbazide 0.5mL after conversion reaction liquid dilution certain multiple, adds 2mL.Boiling water bath 10min, cooling is rapidly surveyed the OD value in 530nm.

Enzyme is lived and defined: in the time of 37 ℃, per minute transforms the enzyme amount that 1 μ mol arginine monohydrochloride generates citrulline.Diacetylmonoxime thiosemicarbazide solution: the 1g Diacetylmonoxime, the 30mg thiosemicarbazide, distilled water is settled to 100mL; Cetyl trimethylammonium bromide solution (CTAB): 1g CTAB, distilled water is settled to 1000mL; Nitration mixture: measure the 70mL strong phosphoric acid, the 160mL vitriol oil slowly is added in the 600mL distilled water, and the cooling back adds the liquor ferri trichloridi of 10mL 10g/L, and adding distil water is settled to 1000mL; L-arginine monohydrochloride-phosphoric acid buffer: 0.5mol/L pH6.0 sodium phosphate buffer 1L adds 0.1mol L-arginine monohydrochloride.

The ADI enzyme gene sequencing of CGMCC 2039 bacterial strains

The chromosomal method of separation and Extraction from this bacterial strain, be adopt known technology (Frederich MA, Roger B, RobertE K, Et al.Short Protocols in Molecular Biology. (4 ^ThEdition) .John Wiley ﹠amp; Sons, Inc., 1999.).The arginine deiminase gene of recombinant plasmid is to increase from total chromosomal DNA by PCR method to obtain.Two primers are according to the arginine deiminase gene order design of the pseudomonas of having reported.Primer-F：ATGTCCGCTGAAAAACAGAAGTACG；Primer-R：TTAGTAGTTGATCGGGTCGCGCACG。Angle the gene of getting enzyme with PCR method, connect cloning vector behind the purifying, transform e. coli jm109, cultivate the back and extract plasmid, purifying, order-checking.

The extraction of ADI

To cultivate 24hr, the distortion pseudomonas of about 250mL (Pseudomonas plecoglossicida) fermented liquid is in the centrifugal 20min → abandoning supernatant of 8000rpm, thalline adds 100mL, pH is 6.0 phosphate buffered saline buffer washing → still centrifugal by above-mentioned condition, and one time → abandoning supernatant of repeated washing again, thalline adds 50mL, pH is 6.0 phosphate buffered saline buffer washing, thalline is broken up → is placed as far as possible the ultrasonic disruption instrument, in 10 ℃, broken 5min under the condition of intensity 10% → with this liquid gets supernatant liquor (about 45ml) in the centrifugal 20min of 12000rpm, preserve in 4 ℃ of refrigerators, stand-by

Detected the ADI enzymic activity of this extracting solution.Get this stand-by supernatant liquor 0.5mL, other adds 5mL 0.2mol/L, pH is arginine monohydrochloride-phosphate buffered saline buffer of 6.0, transform 30min in 37 ℃, with 100 times of its dilutions, get each 2mL of this liquid and pure water (doing blank) respectively in the 10mL colorimetric cylinder, each adds 0.5mL Diacetylmonoxime-thiosemicarbazide solution and 3mL nitration mixture, react 10min in the boiling water bath, sentencing above-mentioned blank in 530nm is that standard is surveyed its OD.The result is 5.1U/mL.

The test of ADI antitumour activity

Place 1640 substratum (adding 10%～20% calf serum) to cultivate in the HepG2 cell strain and the L02 cell of having recovered.Culture condition is 37 ℃, 5%CO ₂Wherein the growth of L02 cell is about 2-3 days; One week of HepG2 cell strain growth.Changed liquid once in 2 days during the cell cultures.

With cultured HepG2 cell strain with as the L02 cell of positive control, add 96 orifice plates, make the cell count in each hole be about 7000, the volume of nutrient solution is every hole 100 μ L.Still cultivate 24hr by above-mentioned condition.

With fermentation and extract the ADI obtain, and,, be made into the different concns gradient, join in 96 orifice plates, make the nutrient solution volume in each hole finally be 200 μ L through 0.2 μ m strainer filtration sterilization as the not inoculation fermentation substratum of negative control.Wherein, in the positive control, each adds the ADI that diluted in the L02 cell culture fluid, and making its ultimate density is 1/10,1/100,1/1000,1/10000; In the negative control, in the HepG2 cell culture fluid, respectively add the blank substratum of 100 μ L; Add the ADI that diluted in the HepG2 cell culture fluid, making its ultimate density is 1/10,1/20,1/40......1/163840,1/327680.Still cultivate 72hr by above-mentioned condition.

With above-mentioned dosing and 96 orifice plates after cultivating 72hr take out, adopt conventional srb assay to measure, survey 540nmOD.

The result shows that ADI has stronger inhibition ability to the growth of HepG2, and this has verified that also HepG2 can not the necessary arginine of self synthetically grown.And for normal liver cell L02, owing to self possess synthetic arginic ability, ADI is just so not strong to its inhibition ability, and this explanation ADI can be used as a kind of medicine of novel treatment liver cancer, is with a wide range of applications.

Beneficial effect

Characteristics of the present invention are distortion pseudomonas CGMCC2039 that screening obtains producing arginine deiminase from a large amount of microorganisms, and it can produce arginine deiminase under the condition of being fit to, and enzyme work is up to more than the 1.6U/ml fermented liquid.

Distortion pseudomonas CGMCC 2039 be a kind of new product arginine deiminase bacterial strain, in the present report both domestic and external, product ADI enzyme Pseudomonas aeruginosa, pseudomonas putida, mycoplasma etc. are arranged.But Pseudomonas aeruginosa is pathogenic bacterium, and pseudomonas putida is a conditioned pathogen, and mycoplasma is that very highly toxic pathogenic agent is arranged, and it is at the ADI of expression in escherichia coli formation inclusion body, and the ADI that therefore recombinates also needs folding again; Be not the report of human pathogenic bacterium at present about the distortion pseudomonas.

We will be out of shape pseudomonas and produce ADI and be used for anti-liver cancer cell HEPG2 experiment, and the result shows that it is very responsive that liver cancer cell HEPG2 produces ADI to this bacterium, illustrate that ADI that this bacterium produces can be used as a kind of medicine of novel treatment liver cancer, is with a wide range of applications.

Below be the embodiment of distortion pseudomonas (Pseudomonas plecoglossicida) CGMCC 2039 screenings, evaluation and fermentative production ADI and ADI Anticancer Activities.But the present invention is not limited to listed several examples.

Embodiment

Embodiment 1

Produce the screening of ADI bacterial classification

From 5 parts of soil samplings such as Wuxi Hui Shan, Xihui Park, Wuxi, canal, Hangzhoupro, capital section, 2 parts in plant materials sample, 2 parts of water samples, 9 duplicate samples altogether.Got behind the sample within 12h strain screening rapidly.Sample is inoculated in the enrichment medium, 30 ℃, 160rpm, behind the cultivation 24hr, dilution, it is dull and stereotyped to coat screening, 30 ℃, behind the cultivation 72hr, selects the bacterium colony of red variable color circle.Choosing 54 strains altogether has the bacterial strain of red variable color circle.Select 18 strains of the big bacterium colony of variable color circle, be seeded in the test tube that the 5mL fermention medium is housed, 30 ℃, 160rpm cultivated 24 hours, adopted Diacetylmonoxime-thiosemicarbazide method that the bacterial strain that primary dcreening operation comes out is carried out the ADI enzyme activity determination, picked out enzyme high bacterial strain alive.

Enrichment medium: L-arginine monohydrochloride 15g/L, vitamin H 3mg/L, VitB1 20mg/L, mixing salt solution 10mL, pH 6.0.

Plate screening substratum: glucose 10g/L, yeast extract paste 2g/L, peptone 2g/L, NaCl 1g/L, L-arginine monohydrochloride 10g/L, phenolsulfonphthalein 0.1g/L, NH ₄Cl 1.5g/L, K ₂HPO ₄1g/L, mixing salt solution 10mL, agar 20g/L, pH 6.0.

Primary dcreening operation fermention medium: glucose 10g/L, yeast extract paste 2g/L, peptone 2g/L, NaCl 1g/L, L-arginine monohydrochloride 10g/L, NH ₄Cl 1.5g/L, K ₂HPO ₄1g/L, mixing salt solution 10mL, pH 7.0.

Wherein: mixing salt solution: MgSO ₄7H ₂O5%, MnSO ₄4H ₂O1%, FeSO ₄7H ₂O0.05%, NaCl0.2%.

Primary dcreening operation sieves again and the results are shown in Table 1, and bacterial strain SW-P1 variable color is irised out existing the fastest, and the variable color number of turns is maximum, and this bacterium source is in the canal water sample.

The multiple sieve result of the bigger phenol red variable color circle bacterial strain of table 118 strain

Numbering	SWP301	SWP302	SWP303	SWP304	SWP305	SWP306	SWP307	SWP308	SWP504
Numbering	SWP301	SWP302	SWP303	SWP304	SWP305	SWP306	SWP307	SWP308	SWP504	Enzyme (OD530nm) alive	0.042	0.020	0.425	0.040	0.267	0.296	0.02	0.028	0.373
Numbering	SWP901	SWP903	SWP905	SWP1	SWP2	SWP309	SWP310	SWP311	SWP902	Enzyme (OD530nm) alive	0.042	0.020	0.425	0.040	0.267	0.296	0.02	0.028	0.373
Numbering	SWP901	SWP903	SWP905	SWP1	SWP2	SWP309	SWP310	SWP311	SWP902	Enzyme (OD530nm) alive	0.517	0.780	0.568	0.955	0.077	0.226	()	0.465	0.540

(annotate: SW is the breadboard numbering of contriver, and P is the pseudomonas code name, and 1～9 is serial number).

Embodiment 2

The evaluation of SW-P1 bacterial strain

The bacterial strain SW-P1 that 1 screening obtains to embodiment is by " uncle's Jie Shi Bacteria Identification handbook (the 8th edition) ", and " common bacteria system identification handbook " carries out physio-biochemical characteristics evaluations (seeing Table 2), and press fine works molecular biology experiment guide method and extract chromosomal DNA, entrust Dalian precious biotechnology company to this bacterium 16S rDNA amplification and order-checking, after obtaining the 16S rDNA sequence of this bacterial strain, on the NCBI website, retrieve the 16S rRNA gene order of relevant bacterial strain among the GenBank, and carry out homology comparison (table 3) with BLAST.Evaluation based on form, physiological and biochemical property and aspects such as 16S rDNA sequence and phylogenetic systematics analysis, bacterial strain SW-P1 is accredited as the distortion pseudomonas, intend called after distortion pseudomonas (Pseudomonasplecoglossicida) SW-P1, this strain bacterium has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No.2039.

Table 2 bacterial strain CGMCC 2039 and the contrast of Rhodopseudomonas part member physio-biochemical characteristics qualification result

Physiological and biochemical property	CGMCC 2039	The distortion pseudomonas	Pseudomonas aeruginosa	Pseudomonas fluorescens	Pseudomonas chlororaphis	Cause golden pseudomonas	P.putida (A)	P.putida (B)	Pseudomonas stutzeri	Pseudomonas mendocina
Physiological and biochemical property	CGMCC 2039	The distortion pseudomonas	Pseudomonas aeruginosa	Pseudomonas fluorescens	Pseudomonas chlororaphis	Cause golden pseudomonas	P.putida (A)	P.putida (B)	Pseudomonas stutzeri	Pseudomonas mendocina	The Gram’s staining flagellum is counted 41 ℃ of growths of 4 ℃ of growths of fluorchrome arginine dihydrolase denitrification gelatin liquefaction egg yolk reaction lipase (Tween 80 hydrolysis) and is utilized: glucosylmannitol D-wood sugar Arabinose D-MANNOSE sucrose trehalose D-malic acid salt tartrate inositol 1B Cit L-Trp nicotine	- ＞1 + - + + - - - - + - - - - - - + - - + - - -	- ＞1 + - - + - - - - + - - - - - - + - - + + - -	- 1 d - + + + + - ± + + - - - - - + - - + d d -	- ＞1 d d - + d + d d + d d d d d d d d d d d d d	- ＞1 + + - + + + + + + + - - + + + d - + d d + -	- ＞1 + + - + - + d d + + - + + d d - - + d d + -	- ＞1 + d - + - - - d + d d d d - - d d - + d - d	- ＞1 d + - + - - - d + d d + d d - d d - d d + +	- 1 - - + - + - - + + d - - d - - - - - - - - -	- 1 - - + + + - - + + - - - d - - - - - - - - -

Annotate: positive more than+90%, negative more than-90%, d 10%-89% is positive

The 16S rDNA sequence (1457bp) of CGMCC 2039 bacterial strains

1 ATTGACGCTG GCGGCAGGCC TAACACATGC AAGTCGAGCG GATGACGGGA GCTTGCTCCT

61 TGATTCAGCG GCGGACGGGT GAGTAATGCC TAGGAATCTG CCTGGTAGTG GGGGACAACG

121 TTCCGAAAGG GGCGCTAATA CCGCATACGT CCTACGGGAG AAAGTGGGGG ATCTTCGGAC

181 CTCACGCTAT CAGATGAGCC TAGGTCGGAT TAGCTAGTTG GTGAGGTAAT GGCTCACCAA

241 GGCGACGATC CGTAACTGGT CTGAGAGGAT GATCAGTCAC ACTGGAACTG AGACACGGTC

301 CAGACTCCTA CGGGAGGCAG CAGTGGGGAA TATTGGACAA TGGGCGAAAG CCTGATCCAG

361 CCATGCCGCG TGTGTGAAGA AGGTCTTCGG ATTGTAAAGC ACTTTAAGTT GGGAGGAAGG

421 GCAGTAAGTT AATACCTTGC TGTTTTGACG TTACCGACAG AATAAGCACC GGCTAACTCT

481 GTGCCAGCAG CCGCGGTAAT ACAGAGGGTG CAAGCGTTAA TCGGAATTAC TGGGCGTAAA

541 GCGCGCGTAG GTGGTTCGTT AAGTTGGATG TGAAAGCCCC GGGCTCAACC TGGGAACTGC

601 ATCCAAAACT GGCGAGCTAG AGTATGGTAG AGGGTGGTGG AATTTCCTGT GTAGCGGTGA

661 AATGCGTAGA TATAGGAAGG AACACCAGTG GCGAAGGCGA CCACCTGGAC TGATACTGAC

721 ACTGAGGTGC GAAAGCGTGG GGAGCAAACA GGATTAGATA CCCTGGTAGT CCACGCCGTA

781 AACGATGTCA ACTAGCCGTT GGAATCCTTG AGATTTTAGT GGCGCAGCTA ACGCATTAAG

841 TTGACCGCCT GGGGAGTACG GCCGCAAGGT TAAAACTCAA ATGAATTGAC GGGGGCCCGC

901 ACAAGCGGTG GAGCATGTGG TTTAATTCGA AGCAACGCGA AGAACCTTAC CAGGCCTTGA

961 CATGCAGAGA ACTTTCCAGA GATGGATGGG TGCCTTCGGG AACTCTGACA CAGGTGCTGC

1021 ATGGCTGTCG TCAGCTCGTG TCGTGAGATG TTGGGTTAAG TCCCGTAACG AGCGCAACCC

1081 TTGTCCTTAG TTACCAGCAC GTCATGGTGG GCACTCTAAG GAGACTGCCG GTGACAAACC

1141 GGAGGAAGGT GGGGATGACG TCAAGTCATC ATGGCCCTTA CGGCCTGGGC TACACACGTG

1201 CTACAATGGT CGGTACAGAG GGTTGCCAAG CCGCGAGGTG GAGCTAATCT CACAAAACCG

1261 ATCGTAGTCC GGATCGCAGT CTGCAACTCG ACTGCGTGAA GTCGGAATCG CTAGTAATCG

1321 CGAATCAGAA TGTCGCGGTG AATACGTTCC CGGGCCTTGT ACACACCGCC CGTCACACCA

1381 TGGGAGTGGG TTGCACCAGA AGTAGCTAGT CTAACCTTCG GGAGGACGGT TACCACGGTG

1441 TGATTCATGA CTGGGGG

Wherein, consist of A25.0%; C22.5%; G31.7%; T20.8%.

Table 3 16SrDNA sequence homology (or similarity) relatively

Strain name	The GenBank numbering	Sequence homology
Strain name	The GenBank numbering	Sequence homology	CGMCC 2039 Pseudomonas plecoglossicida FPC 951 Pseudomonas putida ATCC 12633 Pseudomonas fulva_IAM 1259 Pseudomonas syringae_ATCC 19310 Pseudomonas stutzeri ATCC 17591 Pseudomonas aeruginosa ATCC 10145 Pseudomonas fluorescens IAM 12022 Pseudomonas chlororaphis IAM 12354 Pseudomonas aureofaciens IAM 12353 Pseudomonas mendocina ATCC 25411 Escherichia coli ATCC 25922	AB009457 D84020 D84015 AJ308316 U26261 AF094713 D86001 D84011 D84008 AJ308310 DQ360844	100％ 99.04％ 97.94％ 97.38％ 96.81％ 96.78％ 96.49％ 96.09％ 94.54％ 94.97％ 89.7％ 80.73％

Embodiment 3

The enzymatic production of CGMCC 2039 bacterial strains

Slant culture: press culture medium prescription: peptone 2.5g/L, yeast extract paste 2.5g/L, extractum carnis 2.5g/L, NaCl5g/L, agar 20g, pH7.0.Make the inclined-plane according to a conventional method,, 30 ℃, cultivated 24 hours with CGMCC 2039 inoculation that embodiment 1 screening obtains.

Seed and fermentative medium formula: glucose 15g/L, yeast extract paste 3g/L, peptone 3g/L, L-arginine monohydrochloride 5g/L, Na ₂HPO ₄12H ₂O12g/L, NaH ₂PO ₄2H ₂O3g/L, (NH ₃) ₂HPO ₄2g/L, pH7.0;

Seed culture medium liquid amount 60ml/250ml triangular flask.Connect the cultured slant strains of a ring in triangular flask, 30 ℃, 190 rev/mins, shake-flask culture 14-18 hour.

Adopt 5 liters of fermentor tanks, will be cultured shake-flask seed insert in the fermentor tank, inoculum size is 2%, 30 ℃, 300 rev/mins of rotating speeds, 1: 2 volume/volume of air quantity/minute, under the condition of defoamer 0.02%, cultivated 20 hours; The fermentor cultivation fermentation titer is 1.67U/ml.Table 4 is the fermentor cultivation experimental result.

The enzymatic production of table 4CGMCC 2039 bacterial strains

Fermentation time h	Dissolved oxygen	pH	Cell concentration (g/L)	Enzyme (U/ml) alive	Than enzyme (U/mg) alive
Fermentation time h	Dissolved oxygen	pH	Cell concentration (g/L)	Enzyme (U/ml) alive	Than enzyme (U/mg) alive	0 4 6 8 10 12 14 16 18 20 22 24 26 28 30 42	89.19 0.55 0.26 0.24 0.34 0.29 0.39 9.56 1.00 0.26 1.27 3.79 56.8 60.9 64.5 66.7	6.94 6.54 6.35 6.26 6.26 6.35 6.5 6.59 666 6.76 6.79 686 6.99 7.05 7.13 7.55	/ 0.955 0.855 0.76 2.55 2.31 1.555 1.345 3.395 4.275 4.02 5.16 3.47 3.67 2.665 3.59	/ 0.231 0.387 0.561 0.616 0.723 0.973 1.125 0.818 1.676 0.997 1.328 0.850 0.893 0.754 0.645	/ 0.242 0.453 0.739 0.241 0.313 0.626 0.836 0.241 0.392 0248 0.257 0.245 0.243 0.283 0.180

Embodiment 4

The ADI enzyme gene sequencing of CGMCC 2039 bacterial strains

(1) design of primers and angle with PCR method and to get the enzyme gene and prepare recombinant plasmid

The arginine deiminase gene of recombinant plasmid is to increase from total chromosomal DNA by PCR method to obtain.Two primers are according to the arginine deiminase gene order design of the pseudomonas of having reported.

Primer-F：ATGTCCGCTGAAAAACAGAAGTACG

Primer-R：TTAGTAGTTGATCGGGTCGCGCACG

Method is as follows: chromosomal DNA template 200ng, 2.5 the Tag of unit polysaccharase, the 100nmol primer, 10mmol dNTP1.25uL, final volume 50uL, PCR program: 94 ℃ of sex change 5min, 94 ℃ of sex change 1min of every circulation, 52 ℃ of annealing 1min, 72 ℃ are extended 2min, 10min, totally 30 circulations are extended in last circulation.

We select for use PGEM-T-easy as cloning vector, will angle the enzyme gene of getting to be connected on this carrier, and method is with reference to this carrier operation instruction.

(2) transform screening positive clone

Cultivate e. coli jm109, adopt calcium salt method to prepare competent cell; Adopt the thermal transition method that cloning vector is converted into e. coli jm109, positive colony is picked out in blue white screening.

(3) plasmid extraction, purifying, order-checking

To clone positive bacteria list bacterium colony, insert in the LB/Amp 5ml substratum, 37 ℃, the 200rpm incubated overnight.Collect thalline, extract plasmid, and purifying.Adopting T7 and SP6 is sequencing primer (Primer T7:TAATA CGACT CACTATAGGG; Primer SP6:ATTTA GGTGA CACTA TAGAA), checked order by last sea base health biotechnology company, recording this enzyme gene is 1254bp.

CGMCC 2039 produces arginine deiminase gene order (1254bp)

1 ATGTCCGCTG AAAAACAGAA GTACGGTGTC CACTCCGAAG CAGGCAAGCT GCGCAAGGTA

61 ATGGTCTGCG CTCCGGGACT GGCGCACAAG CGCCTGACCC CGAGCAACTG CGACGAGCTG

121 CTGTTCGACG ATGTGATCTG GGTCGACCAG GCCAAGCGCG ACCACTTCGA CTTCGTCACC

181 AAGATGCGCG AGCGCGGCGT GGATGTGCTG GAAATGCATA ACCTGCTCAC CGACATCGTG

241 CAGAACCCCG AGGCCCTGAA GTGAATCCTC GACCGCAAGA TCACCCCTGA CACCGTCGGG

301 GTGGGCCTGA CCAACGAAGT GCGCAGCTGG CTGGAGGGCC AGGAGCCACG CCACCTCGCC

361 GAGTTCCTGA TCGGCGGCGT GGCCGGCCAG GACCTGCCGG AGAGCGAAGG TGCCAGCGTG

421 GTCAAGATGT ACAACGACTA CCTGGGCCAC TCCAGCTTCA TCCTGCCGCC GCTGCCCAAC

481 ACCCAGTTCA CCCGCGACAC CACCTGCTGG ATCTACGGCG GCGTGACCCT CAACCCGATG

541 TACTGGCCGG CGCGACGCCA GGAAACCCTG CTGACCACCG CCATCTACAA GTTCCACCCC

601 GAGTCCACCA AGGCCGACTT CCAGGTCTGG TACGGCGACC CGGACCAAGA GCACGGCCAG

661 GCCACCCTCG AAGGCGGCGA CGTCATGCCG ATCGGCAAGG GCATCGTGCT GATCGGCATG

721 GGTGAGCGCA CCTCGCGCCA GGCCATCGGC CAACTGGCAC AGAACCTCTT CGCCAAGGGC

781 GCAGTGGAGC AAGTGATCGT CGCCGGGCTG CCGAAGTCCC GTGCGGCCAT GCACCTGGAC

841 ACCGTGTTCA GCTTCTGCGA CCGCGACCTG GTCACGGTTT TCCCGGAAGT GGTGCGCGAG

901 ATCGTGCCGT TCATCATCCG CCCGGACGAA AGCAAGCCCT ACGGCATGGA CGTACGCCGC

961 GAGAACAAGT CGTTCATCGA GGTGGTCGGT GAGCAGCTGG GCGTCAAGCT GCGTGTGGTC

1021 GAGACCGGCG GCAACAGCTT CGCCGCCGAG CGCGAGCAGT GGGATGACGG CAACAACGTG

1081 GTGGCGCTGG AGCCAGGTGT GGTCATCGGC TACGACCGCA ACACCTACAC CAATACCTTG

1141 CTGCGCAAGG CCGGGATAGA GGTCATCACC ATCAGTGCCG GCGAACTGGG CCGGGGCCGT

1201 GGCGGCGGCC ACTGCATGAC CTGCCCGATC GTGCGCGACC CGATCAACTA CTAA

Wherein: 20.4%A; 33.2%C; 31.3%G; 15.1% T;

Embodiment 5

The ADI antitumour activity preliminary experiment of CGMCC 2039 bacterial strains

Used cell

(1) normal cell: L02 (strain of human body normal liver cell)

(2) tumour cell: HepG2 (hepatoma cell strain)

In containing 1640 substratum of 10% calf serum, add blank substratum respectively, and after ultrasonication, be suspended in the distortion pseudomonas preparation of phosphate buffer solution (PBS) as negative control, make said preparation be diluted to finite concentration, add in 37 ℃ of (5%CO ₂) environment in 24 hours the used cell of growth (final every porocyte number is about 7000/300 microlitres), continue to cultivate 72 hours.After cultivating end, the employing srb assay is a cell dyeing, and the 530nm place surveys its OD value on microplate reader.The results are shown in Table 5.

Table 5ADI is to different cell inhibiting rates

Cell strain	L02				HepG2
Cell strain	L02				HepG2					Enzyme (U/mL) inhibiting rate (%) alive	0.5 39.6	0.05 24.3	0.005 2.9	0.0005 9.4	0.043 63.4	0.087 98.0	0.130 96.3	0.173 96.2	0.217 93.1

Show by table 5, use the distortion pseudomonas ADI preparation of test dose among the present invention, caused the suitable obvious suppression of HepG2 tumour cell through 72 hours, normal L02 liver cell is not then made significant difference.

Claims

1. the microorganism strains of arginine deiminase is produced in a strain, the distortion pseudomonas of its minute generic pseudomonadaceae (Pseudomonadaceae) Rhodopseudomonas (Pseudomonas) (Pseudomonas plecoglossicida), preserving number is CGMCC No.2039, and keeps the strain of going down to posterity, the variant of this active this bacterium and the strain of deriving.

2. a microbial fermentation produces the method for arginine deiminase, it is characterized in that:

(1) fermentation strain is CGMCC No.2039;

(2) consisting of of fermention medium: glucose 5-20g/L, yeast extract paste 1-5g/L, peptone 1-5g/L, L-arginine monohydrochloride 5-20g/L, Na ₂HPO ₄12H ₂O 6-15g/L, NaH ₂PO ₄2H ₂O 1-5g/L, (NH ₃) ₂HPO ₄1-5g, pH6.5-8.0;

(3) fermentation culture: the inoculum size by 1%-5% inserts fermention medium with cultured seed, and culture temperature 28-35 ℃, aerobic condition was cultivated 18-28 hour.

3. the ADI gene that is out of shape pseudomonas (Pseudomonas plecoglossicida) CGMCC 2039 according to claim 1 is 1254bp, and its recombinant plasmid PGEM-T-ADI is made up by following method:

(1) separation and Extraction chromosomal DNA from CGMCC 2039 bacterial strains;

(2) from above-mentioned chromosomal DNA, angle the gene of getting enzyme with PCR method;

(3) gene with enzyme is connected on the plasmid PGEM-Teasy.

4. distortion as claimed in claim 1 pseudomonas (Pseudomonas plecoglossicida) CGMCC 2039 and keep the strain of going down to posterity, the variant of this active this bacterium and the application aspect medical of enzyme that the strain of deriving is produced.